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Quantitative proteomics iTRAQ
a
The identification and quantitation of complex protein
mixtures have been facilitated by MS-based quantitative
proteomic techniques. Isobaric tag for relative and
absolute quantification (iTRAQ) consists of amine-specific,
stable isotope reagents that can label peptides of upto
eight different biological samples.
Harini Chandra
Master Layout (Part 1)
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This animation consists of 2 parts:
Part 1: iTRAQ
Part 2: Application of iTRAQ
A
NH
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B
C
NH
Samples
combined
D
4
5
SCX
purification
iTRAQ Label
Protein
samples
NH
Peptide
fragments
Relative abundance
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NH
Trypsin
digestion
LC-MS/MS analysis
m/z
Unwin, R. D. et al., Quantitative Proteomics Analysis Using Isobaric Protein Tags Enables Rapid Comparison of Changes in
Transcript and Protein in Transformed Cells. Mol. Cell. Prot. 2005, 4:924-935.
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Definitions of the components:
Part 1- iTRAQ
1. Isobaric Tag for Relative & Absolute Quantification: iTRAQ is a MS based technique
for relative and absolute quantification of proteins present in up to four cell preparations by
making use of four isobaric, isotope-coded tags that label the proteins via their N-terminal.
2. Protein samples: The samples whose proteins need to
procedure.
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be quantified by iTRAQ
3. Trypsin digestion: Trypsin is a proteolytic enzyme that cleaves proteins at the C-terminal
of arginine and lysine residues except when they are followed by proline. This enables large
protein samples to be broken down in to small peptide fragments.
4. Peptide fragments: The smaller fragments obtained upon cleavage of the protein
samples after trypsin digestion.
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5. iTRAQ label: The iTRAQ reagent consists of a protein reactive group that labels the Nterminus of all peptides as well as free amine groups of lysine side chains, a neutral balance
portion and a reporter group, giving it a total mass of 145. The different distribution of
isotopes between the reporter and balance groups makes the labels isobaric and enables
their detection upon fragmentation and release in MS.
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2
3
4
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Definitions of the components:
Part 1- iTRAQ
6. SCX purification: Tagged peptides are fractionated on a strong cation exchange column to
remove any unbound iTRAQ reagent and to simplify the peptide mixture.
7. LC-MS/MS analysis: The iTRAQ labeled peptides obtained are further purified by reverse
phase liquid chromatography and then analyzed by tandem MS. Each tag releases a distinct
mass reporter ion upon peptide fragmentation, the ratio of which determines the relative
abundances of the peptides.
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Part 1, Step 1:
2
A
3
Trypsin
enzyme
Protein samples
D
C
C-terminal
N-terminal
Trypsin
Digested
fragments
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Action
5
B
As
shown in
the
animatio
n.
Description of the action
First show the four beakers with the different colored ‘protein
samples’ in them. Next show the hand appearing on top of all
the beakers and drops falling from the pipette as shown. The
hand must then disappear and the yellow circle must appear.
The rectangle with dotted line must appear and must be
zoomed into to show the image below. The orange ‘trypsin’
must move across the brown rectangle and must cut it at the
green regions as indicated to give the smaller fragments
below.
Audio Narration
The protein samples to be analyzed are
first digested with trypsin into smaller
peptide fragments. The trypsin cleaves
the proteins at the C-terminal of lysine
and arginine residues unless they are
followed by a proline residue.
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Part 1, Step 2:
O
O
2
N
N
N
3
O
O
H3C
R
117
B 28
N
H
4
R
116
R
115
R
114
B
29
B
30
B
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Action Description of the action
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Show the smaller colored
fragments in the beakers above.
The chemical structure shown
must be added to each of the
beakers to generate the
fragments shown in the beakers
below.
N
H
N
H
A
As
shown in
the
animatio
n.
iTRAQ
labeling
B
N
H
C
D
Audio Narration
The peptide fragments generated are separated by SDS-PAGE to simplify
the mixture and then tagged with the iTRAQ label. The iTRAQ reagent
consists of a reporter group, a balance portion and a peptide reactive group
that interacts with the N-terminus of the peptide or free amino group of
Lysine residues, giving it an overall mass of 145. The reporter group used to
label each peptide sample is unique, with mass varying between 114-117,
thereby enabling the labeling and quantification of four samples
simultaneously. This has been further improved to allow labeling of eight
samples simultaneously.
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Part 1, Step 3:
R
B
N
H
N
H
B
R
2
A
B
C
D
Samples
pooled
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R
B
N
H
4
Action Description of the action
5
As
shown in
the
animatio
n.
Show the four beakers on top
and the bigger empty beaker
below. The contents of the
beaker on top must then be
poured into the beaker below.
Audio Narration
The labeled samples are then pooled together.
B
R
Sample loading
R
B
2
N
H
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Part 1, Step 4:
N
H
Direction of migration
Strong cation
exchange column
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5
As
shown in
the
animatio
n.
Description of the action
Show the contents of the beaker being
poured into the column. They must then
flow through the column in the direction
indicated and be collected in the empty
beaker below.
R
Action
B
N
H
4
Excess iTRAQ label
removed
Audio Narration
The pooled samples are purified on a strong cation
exchange column to remove any excess unbound
iTRAQ reagent. This facilitates sample clean-up
prior to further finer separation and purification
using reverse phase chromatography.
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Part 1, Step 5:
LC-MS/MS analysis
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R
B
N
H
HPLC Pump
Column inlet
from pump
Injector
Mobile phase
3
Sample
LC Column
Sample injector
elution
Sample vials
4
Action
5
Pump Column
As
shown in
the
animatio
n.
Description of the action
Show the beaker on top followed by the arrow. Then show the setup
below with all its labels. The second and third boxes must be
zoomed into to show the figures on the right. The ‘injector’ must
enter the sample bottle with its plunger down. It must remain in this
bottle for a couple of seconds and the plunger must be shown to
move up. This must then move and be injected into the column.
Liquid must be shown to flow through the tube connecting the ‘pump’
and ‘column’. Once the liquid flows, the colour in the column must
change and the liquid must be shown to pass through the tubing at
the outlet.
Column
Column outlet to
detector
Audio Narration
Further purification of the SCX purified
peptides is carried out by reverse phase liquid
chromatography wherein the sample is passed
through a column containing a packed
stationary phase matrix that selectively adsorbs
only certain analyte molecules. The eluted
fractions are further characterized by MS.
LC-MS/MS analysis
Relative quantification
determined by ratio of
peptide pairs.
Relative abundance
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Part 1, Step 6:
2
Detector
m/z
Peptide spectrum
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m2
ESI
m3
Quadrupole
(scanning
mode)
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Action
5
m4
m1
As
shown in
the
animatio
n.
Collision cell
TOF tube
Description of the action
First show all the components of the instrument – the syringe,
four rods, cube, blue rectangle, gray square with the dotted lines
& the detector. Next show appearance of the coloured circles.
Only the red one must move through the rods and after entering
the rectangular box, it must be fragmented to give smaller circles.
These must migrate through the blue tube and get reflected to
reach the ‘detector’. The smallest circles must move the fastest
while the largest must move slowest. Once it reaches the
detector, the graph on top must be shown.
Reflector
Audio Narration
The purified labeled peptide fragments are
then analyzed by MS/MS. The different
masses of the reporter groups allows the
peptide fragments to be identified. The
reporter group is lost during fragmentation.
Relative quantification of up to eight
samples can now be performed using
iTRAQ.
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Part 1, Step 7
MASCOT LC-MS/MS
data analysis
Search title
Sample protein
Enzyme Trypsin
Trypsin
Quantitation iTRAQ
Chymotrypsin
iTRAQ
Peptidase
Taxonomy Bacterial
SILAC
Mammalia
ICAT D8
Carboxymethyl (C)
Fixed
Bacterial
modifications
Plant
Database(s) SwissProt
NCBInr
MSDB
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Variable
modification
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Oxidation (M)
Peptide tol.
1.2
Data file
Data format
Instrument
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Action Description of the
As shown
in
animaion.
Da
# C13
MS/MS tol. 0.2
Monoisotopic
Peptide charge
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Email [email protected]
Your name Proteomics
Da
Average
Choose file
ESI-Q-TOF
Precursor
Start search…
MALDI-TOF
ESI-Q-TOF
action
Audio Narration
MALDI-TOF-TOF
The MS/MS data analysis shareware has some extra inputs such as
First show the computer with the
screen having a form on the inside.
This must be zoomed into and the
form above must be displayed. Each of
the fields must be filled in as shown
with some requiring selection using the
white mouse pointer as depicted.
Quantitation, MS/MS tolerance, peptide charge, instrument etc. in addition to
the fields for PMF. They require inputs from the user regarding the
experimental parameters used such as enzyme cleavage, protein name,
modifications etc. and the desired search criteria like taxonomy, peptide
tolerance etc. Commonly used protein databases against which the MS
information is processed to retrieve sequence data include NCBI, MSDB and
SwissProt. The data file generated from MS is uploaded and the search
carried out.
Master Layout (Part 2)
1
This animation consists of 2 parts:
Part 1: iTRAQ
Part 2: Application of iTRAQ
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3
Serum samples of
normal healthy controls
Immunodepleted
serum
iTRAQ
Immunoaffinity
depletion
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Serum samples of
ovarian cancer patients
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220 unique proteins identified,
14 of which were elevated in
ovarian cancer serum samples.
Boylan, K. L. M. et al., Quantitative proteomic analysis by iTRAQ for the identification of candidate biomarkers in ovarian
cancer serum. Proteome Science 2010, 8:31.
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Definitions of the components:
Part 2- Application of iTRAQ
1. Normal healthy control: Normal healthy control refers to those who do not have the
disease/condition that is being studied. The authors made use of 60 healthy control samples
divided into six groups with ten in each.
2. Ovarian cancer patients: Serum samples from 60 patients with serous ovarian carcinoma
were divided randomly into six groups, with ten patients in each group.
3. Immunoaffinity depletion: Immunoaffinity depletion is a process that is carried out in order
to remove the high abundance proteins present in sera, which hamper the process of
detection of medium or low abundance tumour derived protein markers. In this experiment, the
authors made use of three commercial immunoaffinity depletion methods, the multiple affinity
removal system (MARS), prior to proteomic analysis.
4. Immunodepleted serum: The serum from which the high abundance proteins have been
removed, leaving behind only the medium and low abundance proteins thereby reducing the
dynamic range, is known as immunodepleted serum.
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Part 2, Step 1:
Immunoaffinity
columns
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Serum samples of
normal healthy controls
Immunodepleted serum
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4
Serum samples of
ovarian cancer patients
Action Description of the action
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As
shown in
the
animatio
n.
First show the two tubes on the left with its
components, These components must then be passed
through the first column with pink beads. The blue dots
must be shown to remain in this column and the
remaining components must be passed through the
second column in which the small dark green dots must
remain behind and only the components shown in the
tubes on the right must come out.
Audio Narration
A multiple affinity removal system was made use of to
carry out immunodepletion of the serum samples
from normal controls as well as ovarian cancer
patients. This helped in removing the high abundance
proteins, leaving behind only the medium and low
abundance proteins for iTRAQ analysis.
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Part 2, Step 2:
220 unique proteins identified,
14 of which were elevated in
ovarian cancer serum samples.
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Novel candidate
biomarkers detected
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Action Description of the action
5
As
shown in
the
animatio
n.
First show the two tubes on the left with their
components followed by the arrows showing
the analysis by iTRAQ. Another arrow must
appear from this followed by the graph and
the text boxes as shown.
Proteoglycan-4
Ovarian cancer patients
LPS-binding
protein
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Leu-rich aglycoprotein-1
iTRAQ
analysis
Extracellular
matrix protein-1
Immunodepleted serum
Concentration
Normal healthy controls
Audio Narration
The immunodepleted serum samples were then labeled with
the iTRAQ reagent and analyzed. The authors detected a
total of 220 unique proteins of which 14 were found to be
elevated in the ovarian cancer serum samples compared to
the healthy controls and four novel candidate biomarkers
were detected. Results were validated by Western
immunoblotting.
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Interactivity option 1:Step No: 1 (a)
ADVANTAGES
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LIMITATIONS
2. Majority of proteins identified on
basis of one peptide. Therefore
low confidence of MS/MS data.
3. New search algorithms and
databases needed.
5. Enhanced spectra assignment
will reduce false positives
4. Ability to multiplex
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7.Expanded coverage of proteome
Interacativity Type
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Drag and drop.
1. Quantification of up to eight
samples possible
Options
User must drag and
and drop the seven
statements given
under the heading of
either “advantages”
or “limitations”.
6. Increased precision and
accuracy
Boundary/limits
Results
User has to drag and drop the seven statements
under the heading of either “advantages” or
“limitations”. Statement (1), (4), (6) and (7) must
come under advantages while (2), (3) and (5)
must come under limitations. Everytime the user
drags a statement correctly it must turn green,
otherwise it must turn red and must be returned
back to its original position.
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Questionnaire
1. Which type of chromatography is used for removal of excess iTRAQ reagent prior to LCMS/MS?
Answers: a) Reverse phase b) Gel filtration c) Strong cation exchange d) Affinity
chromatography
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2. The total mass of the iTRAQ reagent is:
Answers: a) 140 b) 143 c) 148 d) 145
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3. Which region of the iTRAQ reagent has variable mass value?
Answers: a) Reporter group b) Peptide-reactive group c) Balance region d) None of the
above
4. A commonly used method for immunodepletion of serum samples:
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Answers: a) Gel filtration b) Multiple affinity removal system c) Ion exchange
chromatography d) SCX
5. Which of the following statements regarding iTRAQ analysis is incorrect?
Answers: a) iTRAQ can label all peptides in up to ten different biological samples
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b) iTRAQ reaction labels the N-terminus of peptides
c) There is no reduction of peptides based on amino acid composition
d) Reporter group is lost during fragmentation
Links for further reading
Research papers:
•
Unwin, R. D. et al., Quantitative Proteomics Analysis Using Isobaric Protein Tags Enables
Rapid Comparison of Changes in Transcript and Protein in Transformed Cells. Mol. Cell.
Prot. 2005, 4:924-935.
•
Wiese, S. et al., Protein labeling by iTRAQ: A new tool for quantitative mass spectrometry
in proteome research. Proteomics 2007, 7: 340-350.
•
Boylan, K. L. M. et al., Quantitative proteomic analysis by iTRAQ for the identification of
candidate biomarkers in ovarian cancer serum. Proteome Science 2010, 8:31.
•
Ye, H. et al., A proteomic approach for plasma biomarker discovery with 8-plex iTRAQ
labeling and SCX-LC-MS/MS. Mol. Cell Biochem. 2010, (epub ahead of print) PMID:
20526653.
•
Glen, A. et al., Eight-plex iTRAQ analysis of variant metastatic human prostate cancer cells
identifies candidate biomarkers of progression: An exploratory study. Prostate 2010, (epub
ahead of print) PMID: 20623638.
•
Hu, H.D. et al., iTRAQ quantitative analysis of multidrug resistance mechanisms in human
gastric cancer cells.